Development Of Monoclonal Antibodies Against Pseudorabies Virus And Double Antibodies Sandwich ELISA For The Virus Detection

Porcine pseudorabies (PR) is an acute infectious disease of domestic animals and wildlife caused by pseudorabies virus (PRV). It was characterized by fever, extreme pruritus and encephalomyelitis. This disease often occurs in the populations of commercial pigs. The infected pregnant sows have the typical symptoms, such as abortion, still born or mummy fetus, and most newborn die shortly after birth. There are serious respiratory symptom and slow-weigth in growing-finisher pigs. It causes great economic losses to pig industry worldwide. Therefore, it is significant important to establish detection methods of PRV on the purpose of control and eradication of pseudorabies.In this study, monoclonal antibodies against PRV were developed, and one method of double antibodies sandwich ELISA(DAS-ELISA) with the rabbit anti-PRV IgGs and a specific monoclonal antibody was established to detect PRV.1. Development of monoclonal antibodies against PRVPRV was passaged and cultivated in PK-15cells, and then purified by sucrose density-gradient ultracentrifugation. Eight-week-old BALB/c mice were subcutaneously injected with purified PRV emulsified with Freund"s complete adjuvant. Mice were subsequently injected two more times with purified PRV mixed with Freund’s incomplete adjuvant at2-weeks intervals. The booster of the double dose of PRV was given at six weeks postimmunization. At3days after last immunization, spleen cells of immunized mice were fused with SP2/0myeloma cell according to the standard procedure of preparation of monoclonal antibody. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen the positive hybridoma cells, and limiting dilution method was performed to subclone these positive hybridoma cells. Four monoclonal antibodies (McAbs) were obtained, named as1A10,2E2,4E6and6B5.The ELISA titers of ascites fluids of1A10and2E2were both1:51200, which belonged to IgG2b subtype, and the titers of ascites of4E6and6B5were both1:12800, which belonged to IgG1subtype. These McAbs had a cross reaction with PRV isolates and vaccine strains. But not other porcine viruses including PPV, PRRSV, PCV-1, PCV-2,and CSFV.2. Development of DAS-ELISA for Detection of PRVThe purified PRV were used as an antigen to obtain the anti-PRV rabbit serum. The ascites of McAbs and rabbit anti-PRV serum were purified by the method of caprylic acid-saturated ammonium sulfate. An double antibodies sandwich ELISA (DAS-ELISA) was established, in which the purified rabbit anti-PRV IgGs were used as a coating antibody and a specific monoclonal antibody as a second antibody. The coating dose of the purified rabbit anti-PRV IgGs was590ng, while dose of monoclonal antibody was123ng. The reaction times for antigens, second antibody and HRP labled antibody were optimized as30min. The coloration time was15min. No cross-reaction was found when PPV, PRPSV, PCV-1, PCV-2and CSFV were tested by this method. The minimum detectable limit of the assay was0.9375μg/mL.80samples of blenna narium of pigs were tested by DAS-ELISA and PCR in parallel. The result showed that the sensitivity of DAS-ELISA was100%and the specificity of DAS-ELISA was91.1%. The coincidence rate between DAS-ELISA and PCR was93.8%.A sensitive and specific sandwich ELISA for detection of PRV was developed. It provided a useful tool for detection of PRV infection.